The so-called diseases of western civilization (chronic conditions such as arthritis, lupus, asthma, and other immune-mediated diseases, osteoporosis, atherosclerosis, other cardiovascular diseases, cancers of the breast, prostate and colon, metabolic syndrome-related conditions such as cardiovascular dysfunctions, diabetes and polycystic ovary syndrome (PCOS), neurodegenerative conditions such as Parkinson's and Alzheimer's, and ophthalmic diseases such as macular degeneration) are now increasingly being viewed as secondary to chronic inflammatory conditions which, in turn, may relate to oxidative stress. A correlation between oxidative stress and processes of aging may explain the rising incidence of these diseases as a direct consequence of an aging population. Lifestyle changes, such as increasingly sedentary habits resulting in the accumulation of fat, may also play a role in the rising significance of oxidative stress-proinflammatory states. A direct link between adiposity and inflammation has recently been demonstrated. Macrophages, potent donors of pro-inflammatory signals, are nominally responsible for this link: Obesity is marked by macrophage accumulation in adipose tissue (Weisberg S P et al [2003] J. Clin Invest 112: 1796-1808) and chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance (Xu H, et al [2003] J. Clin Invest. 112: 1821-1830). Inflammatory cytokine IL-18 is associated with PCOS, insulin resistance and adiposity (Escobar-Morreale H F, et al [2004] J. Clin Endo Metab 89: 806-811). Systemic inflammatory markers such as CRP are associated with unstable carotid plaque, specifically, the presence of macrophages in plaque, which is associated with instability can lead to the development of an ischemic event (Alvarez Garcia B et al [2003] J Vasc Surg 38: 1018-1024). There are documented cross-relationships between these risk factors. For example, there is higher than normal cardiovascular risk in patients with rheumatoid arthritis (RA) (Dessein P H et al [2002] Arthritis Res. 4: R5) and elevated C-peptide (insulin resistance) is associated with increased risk of colorectal cancer (Ma J et al [2004] J. Natl Cancer Inst 96:546-553) and breast cancer (Malin A. et al [2004] Cancer 100: 694-700). The genesis of macrophage involvement with diseased tissues is not yet fully understood, though various theories postulating the “triggering” effect of some secondary challenge (such as viral infection) have been advanced. What is observed is vigorous crosstalk between macrophages, T-cells, and resident cell types at the sites of disease. For example, the direct relationship of macrophages to tumor progression has been documented. In many solid tumor types, the abundance of macrophages is correlated with prognosis (Lin E Y and Pollard J W [2004] Novartis Found Symp 256: 158-168). Reduced macrophage population levels are associated with prostate tumor progression (Yang G et al [2004] Cancer Res 64:2076-2082) and the “tumor-like behavior of rheumatoid synovium” has also been noted (Firestein G S [2003] Nature 423: 356-361). At sites of inflammation, macrophages elaborate cytokines such as interleukin-1-beta and interleukin-6.
A ubiquitous observation in chronic inflammatory stress is the up-regulation of heat shock proteins (HSP) at the site of inflammation, followed by macrophage infiltration, oxidative stress and the elaboration of cytokines leading to stimulation of growth of local cell types. For example, this has been observed with unilateral obstructed kidneys, where the sequence results in tubulointerstitial fibrosis and is related to increases in HSP70 in human patients (Valles, P. et al [2003] Pediatr Nephrol. 18: 527-535). HSP70 is required for the survival of cancer cells (Nylandsted J et al [2000] Ann NY Acad Sci 926: 122-125). Eradication of glioblastoma, breast and colon xenografts by HSP70 depletion has been demonstrated (Nylansted J et al [2002] Cancer Res 62:7139-7142; Rashmi R et al [2004] Carcinogenesis 25: 179-187) and blocking HSF1 by expressing a dominant-negative mutant suppresses growth of a breast cancer cell line (Wang J H et al [2002] BBRC 290: 1454-1461). It is hypothesized that stress-induced extracellular HSP72 promotes immune responses and host defense systems. In vitro, rat macrophages are stimulated by HSP72, elevating NO, TNF-alpha, IL-1-beta and IL-6 (Campisi J et al [2003] Cell Stress Chaperones 8: 272-86). Significantly higher levels of (presumably secreted) HSP70 were found in the sera of patients with acute infection compared to healthy subjects and these levels correlated with levels of IL-6, TNF-alpha, IL-10 (Njemini R et al [2003] Scand. J. Immunol 58: 664-669). HSP70 is postulated to maintain the inflammatory state in asthma by stimulating pro-inflammatory cytokine production from macrophages (Harkins M S et al [2003] Ann Allergy Asthma Immunol 91: 567-574). In esophageal carcinoma, lymph node metastasis is associated with reduction in both macrophage populations and HSP70 expression (Noguchi T. et al [2003] Oncol. 10: 1161-1164). HSPs are a possible trigger for autoimmunity (Purcell A W et al [2003] Clin Exp Immunol. 132: 193-200). There is aberrant extracellular expression of HSP70 in rheumatoid joints (Martin C A et al [2003] J. Immunol 171: 5736-5742). Even heterologous HSPs can modulate macrophage behavior: H. pylori HSP60 mediates IL-6 production by macrophages in chronically inflamed gastric tissues (Gobert A P et al [2004] J. Biol. Chem 279: 245-250).
In addition to immunological stress, a variety of environmental conditions can trigger cellular stress programs. For example, heat shock (thermal stress), anoxia, high osmotic conditions, hyperglycemia, nutritional stress, endoplasmic reticulum (ER) stress and oxidative stress each can generate cellular responses, often involving the induction of stress proteins such as HSP70.
One common feature of nearly all of the emerging diseases in the Western world is the complexity of the underlying biochemical dysfunctions. New methodology for identifying the core biochemical lesions in disease conditions is needed. Such methodology would provide a first step to the development of predictive diagnostics and adequately targeted interventions.
About 40,000 women die annually from metastatic breast cancer in the U.S. Current interventions focus on the use of chemotherapeutic and biological agents to treat disseminated disease, but these treatments almost invariably fail in time. At earlier stages of the disease, treatment is demonstrably more successful: systemic adjuvant therapy has been studied in more than 400 randomized clinical trials, and has proven to reduce rates of recurrence and death more than 15 years after treatment (Hortobagyi G N. (1998) N Engl J Med. 339 (14): 974-984). The same studies have shown that combinations of drugs are more effective than just one drug alone for breast cancer treatment. However, such treatments significantly lower the patient's quality of life, and have limited efficacy. Moreover, they may not address slow-replicating tumor reservoirs that could serve as the source of subsequent disease recurrence and metastasis. A successful approach to the treatment of recurrent metastatic disease must address the genetic heterogeneity of the diseased cell population by simultaneously targeting multiple mechanisms of the disease such as dysregulated growth rates and enhanced survival from (a) up-regulated stress-coping and anti-apoptotic mechanisms, and (b) dispersion to sequestered and privileged sites such as spleen and bone marrow. Cellular diversification, which leads to metastasis, produces both rapid and slow growing cells. Slow-growing disseminated cancer cells may differ from normal cells in that they are located outside their ‘normal’ tissue context and may up-regulate both anti-apoptotic and stress-coping survival mechanisms. Global comparison of cancer cells to their normal counterparts reveals underlying distinctions in system logic. Cancer cells display up-regulated stress-coping and anti-apoptotic mechanisms (e.g. NF-kappa-B, Hsp-70, MDM2, survivin etc.) to successfully evade cell death (Chong Y P, et al. (2005) Growth Factors. September; 23 (3): 233-44; Rao R D, et al (2005) Neoplasia. October; 7 (10): 921-9; Nebbioso A, et al (2005) Nat Med. January; 11 (1): 77-84). Many tumor types contain high concentrations of heat-shock proteins (HSP) of the HSP27, HSP70, and HSP90 families compared with adjacent normal tissues (Ciocca at al 1993; Yano et al 1999; Cornford at al 2000; Strik et al 2000; Ricaniadis et al 2001; Ciocca and Vargas-Roig 2002). The role of HSPs in tumor development may be related to their function in the development of tolerance to stress (Li and Hahn 1981) and high levels of HSP expression seem to be a factor in tumor pathogenesis. Among other mechanisms individual HSPs can block pathways of apoptosis (Volloch and Sherman 1999). Studies show HSP70 is required for the survival of cancer cells (Nylandsted J, Brand K, Jaattela M. (2000) Ann N Y Acad Sci. 926: 122-125). Eradication of glioblastoma, breast and colon xenografts by HSP70 depletion has been demonstrated, but the same treatment had no effect on the survival or growth of fetal fibroblasts or non-tumorigenic epithelial cells of breast (Nylandsted J, et al (2002) Cancer Res. 62 (24): 7139-7142; Rashmi R, Kumar S, Karunagaran D. (2004) Carcinogenesis. 25 (2): 179-187; Barnes J A, et al. (2001) Cell Stress Chaperones. 6 (4): 316-325) and blocking HSF1 by expressing a dominant-negative mutant suppresses growth of a breast cancer cell line (Wang J H, et al. (2002) Biochem Biophys Res Commun. 290 (5): 1454-1461). Stress can also activate the nuclear factor kappa B (NF-kappa B) transcription factor family. NF-kappa-B is a central regulator of the inflammation response that regulates the expression of anti-apoptotic genes, such as cyclooxygenases (COX) and metalloproteinases (MMPs), thereby favoring tumor cell proliferation and dissemination. NF-kappa-B can be successfully inhibited by peptides interfering with its intracellular transport and/or stability (Butt A J, et al. (2005) Endocrinology. July; 146 (7): 3113-22). Human survivin, an inhibitor of apoptosis, is highly expressed in various tumors (Ambrosini G, Adida C, Altieri D C. (1997) Nat. Med. 3 (8): 917-921) aberrantly prolonging cell viability and contributing to cancer. It has been shown that ectopic expression of survivin can protect cells against apoptosis (Li F, et al. (1999) Nat. Cell Biol. 1 (8): 461-466). Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. Peptides modeled on the MDM2-binding pocket of p53 can inhibit the negative feedback of MDM2 on p53 commonly observed in cancer cells (Midgley C A, et al. (2000) Oncogene. May 4; 19 (19): 2312-23; Zhang R, et al. (2004) Anal Biochem. August 1; 331 (1): 138-46). The role of protein degradation rates and the proteasome in disease has recently come to light Inhibitors of HSP90 (a key component of protein degradation complexes) such as bortezomib are in clinical testing and show promise as cancer therapeutics (Mitsiades C S, et al. 2006 Curr Drug Targets. 7(10):1341-1347). A C-terminal metal-binding domain (MBD) of insulin-like growth factor binding protein-3 (IGFBP-3) can rapidly (<10 min) mobilize large proteins from the extracellular milieu into the nuclei of target cells (Singh B K, et al. (2004) J Biol Chem. 279: 477-487). Here we extend these observations to show that MBD is a systemic ‘guidance system’ that attaches to the surface of red blood cells and can mediate rapid intracellular transport of its ‘payload’ into the cytoplasm and nucleus of target cells at privileged sites such as spleen and bone marrow in vivo. The amino acid sequence of these MBD peptides can be extended to include domains known to inhibit HSP, survivin, NF-kappa-B, proteasome and other intracellular mechanisms. The MBD mediates transport to privileged tissues and intracellular locations (such as the nucleus) in the target tissue. In this study we ask whether such MBD-tagged peptides might act as biological modifiers to selectively enhance the efficacy of existing treatment modalities against cancer cells. Patients presenting with metastatic disease generally face a poor prognosis. The median survival from the time of initial diagnosis of bone metastasis is 2 years with only 20% surviving 5 years (Antman et al. (1999) JAMA.; 282: 1701-1703; Lipton A. (2005) North American Pharmacotherapy: 109-112). A successful systemic treatment for recurrent metastatic disease is the primary unmet medical need in cancer.
Part of the lack of success in treating metastatic disease may have to do with a lack of understanding of the metastatic disease process. Unlike the primary tumor event, which is primarily a dysfunction of unregulated growth, metastatic cells must generally adapt to unusual environments in body locations that are distant to the original tumor site. Thus, most traditional interventions designed to treat a primary tumor, which focus on controlling tumor cell growth, may be fundamentally unsuited to the treatment of metastatic disease, which is a disease of adaptation. Thus there is a need for identifying the biochemical correlates of cellular adaptivity.
Diabetes is a rapidly expanding epidemic in industrial societies. The disease is caused by the body's progressive inability to manage glucose metabolism appropriately. Insulin production by pancreatic islet cells is a highly regulated process that is essential for the body's management of carbohydrate metabolism. The primary economic and social damage of diabetes is from secondary complications that arise in the body after prolonged exposure to elevated blood sugar. These include cardiovascular complications, kidney disease and retinopathies. Most interventions so far developed for diabetic conditions focus on controlling blood sugar, the primary cause of subsequent complications. However, despite the availability of several agents for glycemic control, the population of individuals with poorly controlled blood sugar continues to explode. 40% of kidney failure is currently associated with diabetes, and that percentage is expected to rise.
One potential approach to treating the complications of diabetes is to focus on the cellular biochemistry of organs that are particularly sensitive to elevated blood sugar levels. Advanced glycosylation end products of proteins (AGEs) are non-enzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. Cellular actions of advanced glycation end-products (AGE) are mediated by a receptor for AGE (RAGE), a novel integral membrane protein (Neeper M et al [1992] J. Biol. Chem. 267: 14998-15004). Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The bioactivity of RAGE is governed by the settings in which these ligands accumulate, such as diabetes, inflammation and tumors. Vascular complications of diabetes such as nephropathy, cardiomyopathy and retinopathy, may be driven in part by the AGE-RAGE system (Wautier J-L, et al [1994] Proc. Nat. Acad. Sci. 91: 7742-7746; Barile G R et al [2005] Invest. Ophthalm. Vis. Sci. 46: 2916-2924; Yonekura H et al [2005] J. Pharmacol. Sci. 97: 305-311). Specific downstream cellular molecular events are now believed to mediate some of the damaging consequences of RAGE activation, and generate a rationale for chemical, biological and genetic interventions in these types of hypertrophic disease processes (Cohen M P et al [2005] Kidney Int. 68: 1554-1561; Cohen M P et al [2002] Kidney Int. 61: 2025-2032; Wendt T et al [2006] Atherosclerosis 185: 70-77; Wolf G et al [2005] Kidney Int. 68: 1583-1589). Soluble RAGE is associated with albuminuria in human diabetics (Humpert P M et al [2007] Cardiovasc. Diabetol. 6: 9) and in animal models of diabetic nephropathy such as the db/db mouse (Yamagishi S et al [2006] Curr. Drug Discov. Technol. 3: 83-88; Sharma K et al [2003] Am J. Physiol. Renal Physiol. 284: F1138-F1144). In the complex disease process of diabetic progression the causal interplay of hypertensive, glycemic, inflammatory and endocrinological factors is difficult to parse. Nevertheless, magnetic resonance imaging of the db/db mouse reveals progressive cardiomyopathic changes as diabetes progresses. Relatively early in the disease process (9 weeks), left ventricular hypertrophy (LVH) is observed. In human populations, LVH correlates with elevated levels of NT-pro-BNP and cardiac Troponin T (cTnT) in serum (Arteaga E et al [2005] Am Heart J. 150: 1228-1232; Lowbeer C et al [2004] Scand J. Clin. Lab Invest. 64: 667-676).
TOR (target of rapamycin) proteins are conserved Ser/Thr kinases found in diverse eukaryotes ranging from yeast to mammals. The TOR kinase is found in two biochemically and functionally distinct complexes, termed TORC1 and TORC2. mTORC1 contains mTOR phosphorylated predominantly on S2448, whereas mTORC2 contains mTOR phosphorylated predominantly on S2481 (Copp J et al [2009] Cancer Res. 69: 1821-1827). Aided by the compound rapamycin, which specifically inhibits TORC1, the role of TORC1 in regulating translation and cellular growth has been extensively studied. mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton (Jacinto E, et al [2004] Nat Cell Biol. 6: 1122-1128). The physiological roles of TORC2 have remained largely elusive due to the lack of pharmacological inhibitors and its genetic lethality in mammals. PRR5 and related proteins are a new class of molecules found in association to mTORC2 complex, and may be required cofactors for the function of this central regulator of cellular biochemistry. The PRR5 gene encodes a conserved proline-rich protein predominant in kidney (Johnstone C N et al [2005] Genomics 85: 338-351). The PRR5 class of proteins is believed to physically associate with mTORC2 and regulate aspects of growth factor signaling and apoptosis (Woo S Y et al [2007] J. Biol. Chem. 282: 25604-25612; Pearce L R et al [2007] Biochem J. 405: 513-522; Thedieck K et al [2007] PLoS ONE 2: e1217). In this invention, the importance of a particular domain within PRR5 (referred to as the PRR5D sequence) comprising the sequence HESRGVTEDYLRLETLVQKVVSPYLGTYGL (SEQ ID NO:3) is demonstrated. This sequence is conserved in human PRR5 isoforms and PRR5L as well as in rat and mouse. Other obligate partners of Rictor, a central defining protein component of the mTORC2 complex, include Sin1 (also known as MIP1). Sin1 is an essential component of TORC2 but not of TORC1, and functions similarly to Rictor, the defining member of TORC2, in complex formation and kinase activity. Knockdown of Sin1 decreases Akt phosphorylation in both Drosophila and mammalian cells and diminishes Akt function in vivo. It also disrupts the interaction between Rictor and mTOR. Furthermore, Sin1 is required for TORC2 kinase activity in vitro (Yang Q et al [2006] Genes Dev. 20: 2820-2832). mTOR, SIN1 and Rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt, SGK1 (serum- and glucocorticoid-induced protein kinase 1), and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation (Facchinetti V et al [2008] EMBO J. 27: 1932-1943; García-Martínez J M and Alessi D R [2008] Biochem J. 416: 375-385; Jacinto E and Lorberg A. [2008] Biochem J. 410:19-37).
mTORC2 activity was elevated in glioma cell lines as well as in primary tumor cells as compared with normal brain tissue (Masri J et al [2007] Cancer Res. 67: 11712-11720). In these lines Rictor protein and mRNA levels were also elevated and correlated with increased mTORC2 activity. Protein kinase C alpha (PKC alpha) activity was shown to be elevated in rictor-overexpressing lines but reduced in rictor-knockdown clones, consistent with the known regulation of actin organization by mTORC2 via PKC alpha. Xenograft studies using these cell lines also supported a role for increased mTORC2 activity in tumorigenesis and enhanced tumor growth. These data suggest that mTORC2 is hyperactivated in gliomas and functions in promoting tumor cell proliferation and invasive potential. mTORC2 and its activation of downstream AGC kinases such as PKC-alpha, SGK1 and Akt have also been implicated in cancers of the prostate and breast (Guertin D A et al [2009] Cancer Cell. 15: 148-159; Sahoo S et al [2005] Eur J Cancer. 41: 2754-2759; Guo J, et al [2008] Cancer Res. 68: 8473-8481).
IRS-1 and IRS-2 are master traffic regulators in intracellular signal transduction pathways associated with growth and metabolism, playing key roles in the docking of accessory proteins to phosphorylated insulin and IGF receptors. Although similar in function, activated IRS-1 and IRS-2 proteins generate subtly different cellular outcomes, at least in part through the phosphorylation of different Akt (especially Akt 1 and Akt 2) and MAP kinase isoforms.
The significance of IRS-2 to IRS-1 ratios in inflammatory disease processes has never been explicitly cited. The possibility of using specific modulators of the IRS-2:IRS-1 to intervene in such disease processes has not been explicitly proposed. Such modulators might include, for example, treatments or compounds that preferentially reduce IRS-2 (versus IRS-1) signaling, or preferentially increase IRS-1 (versus IRS-2) signaling. Some unrelated observations of potential significance here are the use of a KRLB domain-specific inhibitor for IRS-2, the use of selected HIV protease inhibitors such as nelfinavir, saquinavir and ritonavir (previously shown to selectively inhibit IRS-2 over IRS-1). In this invention, the modulating effects of certain peptides such as humanin, PRR5 domain (PRR5D), and NPKC on IRS-2 versus IRS-1, both in vitro and in vivo, are described. The specific induction of IRS-2 in human kidney cells by a ligand of RAGE, first demonstrated here, and the modulation of that induction by humanin and NPKC peptides, further suggests the involvement of similar mechanisms of pathology in other RAGE-related proliferative or inflammatory conditions such as metastatic breast cancer, Alzheimer's disease, atherosclerosis, other cardiovascular conditions, arthritis, other autoimmune conditions and sepsis. Also shown here for the first time is the direct correlation between kidney IRS-2 levels, kidney collagen-IV levels and kidney function in diabetic db/db mice. Other peptides may also modulate IRS-2:IRS-1 ratios, including but not limited to MBD-KRLB (SEQ ID NO:3).
IRS-1 and IRS-2 are expressed in normal mammary epithelial cells and in breast carcinoma cells, where they have been implicated in mediating signals to promote tumor cell survival, growth and motility. Although IRS-1 and IRS-2 are homologous, some recent studies have revealed distinct functions for these adaptor proteins in regulating breast cancer progression. Specifically, IRS-2 is a positive regulator of metastasis, whereas IRS-1 may be a suppressor of metastasis and cell motility (Gibson S L et al [2007] Cell Cycle. 6: 631-637; Jackson J G et al [2001] Oncogene. 20: 7318-7325; Ibrahim Y H et al [2008] Mol Cancer Res. 6: 1491-1498). Other studies suggest that both IRS-1 and IRS-2 can promote metastasis (Dearth R K, et al [2006] Mol Cell Biol. 26: 9302-9314).
We have recently shown (Singh B K and Mascarenhas D D [2008] Am J Nephrol. 28: 890-899) that wild type humanin and other peptides can reduce albuminuria in db/db mice. The accompanying biochemical changes in mouse kidney tissue, as well as in cell culture systems using human kidney cells stimulated with glycated hemoglobin, suggest a tight correlation of albuminuria with elevated IRS-2 levels, higher PKC-alpha/beta phosphorylation and changes in Akt status. We now show that SGK1 is also elevated in diabetic kidneys. Upon treatment with humanin and related peptides, the perturbations in these markers are simultaneously ameliorated. Moreover, we show here for the first time that treatment with nephrilin, a peptide designed to compete with the PRR5-Rictor interface, also reduced albuminuria, phospho-PKC, IRS-2 and SGK1 in diabetic kidneys. The RAGE system has been implicated in cancer and metastasis (Logsdon C D et al [2007] Curr Mol Med. 7: 777-789). In our ELISA assays of extracts prepared from paired cell lines (each pair from a single patient) we have demonstrated for the first time that primary tumor cells differed from metastatic variants by virtue of the metastatic variants (but not the primary tumor cells) being able to dramatically enhance levels of IRS-2, phospho-PKC-alpha/beta and Akt status in response to glycated hemoglobin (RAGE ligand) stimulus. We also showed that breast cancer cells that successfully set up liver metastasis in mice have significantly elevated IRS2:IRS1 ratios compared to the original cultured human cancer cell line used in the experiment. Taken together with the observation made here for the first time that there is a physical association between Rictor and IRS proteins in kidney cells, and that this association is significantly reduced by treatment with nephrilin peptide (which reduces albuminuria in db/db mice) we propose a fundamentally new insight into the mechanism of key disease processes such as diabetes and cancer metastasis, and diseases involving a systemic inflammatory component. We further propose new intervention strategies for treating these disease processes. Specifically, we propose criteria for recognizing a global, disease-associated cellular biochemical signature characterized by distinctively altered (a) isotype levels, cellular location and phosphorylation status of IRS proteins (b) ratios of active mTORC2 to mTORC1; or (c) isotype levels and phosphorylation status of AGC family kinases such as Akt, SGK and PKC (for example, levels of SGK1 and Akt2, and phosphorylation of PKC-alpha/beta). These factors, taken together, constitute a signature of a global cellular response to stress, such as inflammatory stress mediated by the RAGE system.
In diabetic humans and db/db mice the receptor for advanced glycated end products (RAGE) is activated by systemic ligands such as amphoterin and glycated hemoglobin (Goldin A et al [2006] Circulation 114: 597-605). RAGE has been implicated in the development of kidney dysfunction consequent to elevated blood sugar (Tan A L et al [2007] Semin. Nephrol. 27:130-143). The intracellular biochemical events downstream of RAGE activation leading to the loss of kidney function and albuminuria in db/db mice are not well understood. RAGE blockade through the use of soluble RAGE decoys has been proposed as a method for controlling complications of diabetes in humans (Yamagishi S et al [2007] Curr. Drug Targets 8:1138-1143; Koyama H et al [2007] Mol Med 13:625-635). Kidney mesangial cell matrix expansion characterized by excessive deposition of collagen-IV and fibronectin is an often-cited correlate of disease progression (Tsilibary E C et al [2003] J. Pathol. 200: 537-546). However, effective interventions based on this hypothesis have yet to be developed. Recently, the inhibition of protein kinase C (PKC) isoforms has been proposed as a possible therapeutic intervention for kidney disease (Tuttle K R et al [2003] Am. J. Kidney Dis. 42: 456-465). A peptide capable of inhibiting PKC beta II in cultured cells has been described (Ron D et al [1995] J. Biol. Chem. 270: 24180-24187). Correlation matrices or dendograms (Peterson L E [2003] Comput. Methods Programs Biomed. 70: 107-119) constructed from RAGE-adaptive datasets gathered in cultured kidney cell and kidney tissue extracts can help identify reliable biochemical correlates of disease, and can guide the development of effective therapeutic interventions. Although correlations do not reveal causative links, the clustering of biochemical correlates can help define ‘virtual regulons’ around which hypothesis-driven interventions can be designed and tested. This invention describes methods for surveying a panel of intracellular biochemical readouts in cultured human embryonic kidney (HEK) 293 cells challenged with glycated hemoglobin and various chemical and peptide inhibitors. From these data a method is described for selecting a subset of readouts that are significantly impacted by RAGE ligand in these cells. Taken together, these readouts are referred to as an “adaptive signature”. In this context, RAGE ligand is referred to as a “provocative agent” for the derivation of adaptive signatures. A provocative agent is a chemical or biological substance that provokes a change in cellular signaling that resembles, at least in part, the changes seen in a disease condition. Adaptive signature refers to the delta, or difference in readouts, between cells that are treated with a specific provocative agent and cells that are treated with control, such as saline. Similar methodology can be applied to tissues from animals or humans that have been exposed to varying levels of a provocative agent. As an example, kidney extracts from albuminuric db/db mice can be assayed for these selected biochemical markers and compared with a group of control animals who have not developed albuminuria. Correlation matrices constructed from these data can subsequently suggest possible modifications to our current understanding of diabetic kidney disease, based on the adaptive signatures revealed. Statistical clustering of deltas, suggesting common regulation, can be used to assign “virtual regulons.” Three key features of this methodology are (a) the choice of provocative agent (b) the use of delta values as opposed to the more traditional approach of using actual biochemical assay values in profiling, and (c) the use of correlation matrices or dendograms to generate virtual regulon clusters based on related adaptive response, rather than logical pathway analysis.
Despite the worldwide epidemic of chronic kidney disease complicating diabetes mellitus, current therapies directed against nephroprogression are limited to angiotensin conversion or receptor blockade. Nonetheless, additional therapeutic possibilities are slowly emerging. The diversity of therapies currently in development reflects the pathogenic complexity of diabetic nephropathy. The three most important candidate drugs currently in development include a glycosaminoglycan, a protein kinase C (PKC) inhibitor and an inhibitor of advanced glycation (Williams M E [2006] Drugs. 66: 2287-2298). Treatment of hypertrophic conditions of the heart and kidney using protein kinase C-beta inhibitors (Koya D et al [2000] FASEB J. 14: 439-447) represents an alternative to RAGE blockade and TGF-beta-1 blockade approaches to new interventions in hypertrophic disease states.
Renal failure characterized by proteinuria and mesangial cell expansion is observed in a number of non-diabetic states. Many forms of renal disease that progress to renal failure are characterized histologically by mesangial cell proliferation and accumulation of mesangial matrix. These diseases include IgA nephropathy and lupus nephritis. Bone marrow transplantation (BMT) is an effective therapeutic strategy for leukemic malignancies and depressed bone marrow following cancer. However, its side effects on kidneys have been reported. (Otani M et al [2005] Nephrology 10: 530-536). Some hematological malignancies associated with nephrotic syndrome include Hodgkin's and non-Hodgkin's lymphomas and chronic lymphocytic leukemia (Levi I [2002] Lymphoma. 43: 1133-1136). Cancer drugs such as mitomycin, cisplatin, bleomycin, and gemcitabine (Saif M W and McGee P J [2005] JOP. 6: 369-374) and the anti-angiogenic agent bevacizumab (Avastin) (Gordon M S and Cunningham D [2005] Oncology. 69 Suppl 3: 25-33) and irradiation are also suggested to be nephrotoxic. Moreover, the observed cardiotoxicity of drugs such a 5-fluorouracil and capecitabine may be secondary to renal toxicity of these drugs (Jensen S A and Sorensen J B [2006] Cancer Chemother Pharmacol. 58: 487-493). There are a large number of glomerular diseases that may be responsible for a nephrotic syndrome, the most frequent in childhood being minimal change disease. Denys-Drash syndrome and Frasier syndrome are related diseases caused by mutations in the WT1 gene. Familial forms of idiopathic nephrotic syndrome with focal and segmental glomerular sclerosis/hyalinosis have been identified with an autosomal dominant or recessive mode of inheritance and linkage analysis have allowed to localize several genes on chromosomes 1, 11 and 17. The gene responsible for the Finnish type congenital nephrotic syndrome has been identified. This gene, named NPHS1, codes for nephrin, which is located at the slit diaphragm of the glomerular podocytes and is thought to play an essential role in the normal glomerular filtration barrier (Salomon R et al [2000] Curr. Opin. Pediatr. 12: 129-134).
Familial mutations in parkin gene are associated with early-onset PD. Parkinson's disease (PD) is characterized by the selective degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). A combination of genetic and environmental factors contributes to such a specific loss, which is characterized by the accumulation of misfolded protein within dopaminergic neurons. Among the five PD-linked genes identified so far, parkin, a 52 kD protein-ubiquitin E3 ligase, appears to be the most prevalent genetic factor in PD. Mutations in parkin cause autosomal recessive juvenile parkinsonism (AR-JP). The current therapy for Parkinson's disease is aimed to replace the lost transmitter, dopamine. But the ultimate objective in neurodegenerative therapy is the functional restoration and/or cessation of progression of neuronal loss (Jiang H, et al [2004] Hum Mol Genet. 13 (16): 1745-54; Muqit M M, et al [2004] Hum Mol Genet. 13 (1): 117-135; Goldberg M S, et al [2003] J Biol Chem. 278 (44): 43628-43635). Over-expressed parkin protein alleviates PD pathology in experimental systems. Recent molecular dissection of the genetic requirements for hypoxia, excitotoxicity and death in models of Alzheimer disease, polyglutamine-expansion disorders, Parkinson disease and more, is providing mechanistic insights into neurotoxicity and suggesting new therapeutic interventions. An emerging theme is that neuronal crises of distinct origins might converge to disrupt common cellular functions, such as protein folding and turnover (Driscoll M, and Gerstbrein B. [2003] Nat Rev Genet. 4(3): 181-194). In PC12 cells, neuronally differentiated by nerve growth factor, parkin overproduction protected against cell death mediated by ceramide Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of parkin. (Darios F. et al [2003] Hum Mol Genet. 12 (5): 517-526). Overexpressed parkin suppresses toxicity induced by mutant (A53T) and wt alpha-synuclein in SHSY-5Y cells (Oluwatosin-Chigbu Y. et al [2003] Biochem Biophys Res Commun. 309 (3): 679-684) and also reverses synucleinopathies in invertebrates (Haywood A F and Staveley B E. [2004] BMC Neurosci. 5(1): 14) and rodents (Yamada M, Mizuno Y, Mochizuki H. (2005) Parkin gene therapy for alpha-synucleinopathy: a rat model of Parkinson's disease. Hum Gene Ther. 16(2): 262-270; Lo Bianco C. et al [2004] Proc Natl Acad Sci USA. 101(50): 17510-17515). On the other hand, a recent report claims that parkin-deficient mice are not themselves a robust model for the disease (Perez F A and Palmiter R D [2005] Proc Natl Acad Sci USA. 102 (6): 2174-2179). Nevertheless, parkin therapy has been suggested for PD (Butcher J. [2005] Lancet Neurol. 4(2): 82).
Variability within patient populations creates numerous problems for medical treatment. Without reliable means for determining which individuals will respond to a given treatment, physicians are forced to resort to trial and error. Because not all patients will respond to a given therapy, the trial and error approach means that some portion of the patients must suffer the side effects (as well as the economic costs) of a treatment that is not effective in that patient.
For some therapeutics targeted to specific molecules within the body, screening to determine eligibility for the treatment is routinely performed. For example, the estrogen antagonist tamoxifen targets the estrogen receptor, so it is normal practice to only administer tamoxifen to those patients whose tumors express the estrogen receptor. Likewise, the anti-tumor agent trastuzumab (HERCEPTIN®) acts by binding to a cell surface molecule known as HER2/neu; patients with HER2/neu negative tumors are not normally eligible for treatment with trastuzumab. Methods for predicting whether a patient will respond to treatment with IGF-I/IGFBP-3 complex have also been disclosed (U.S. Pat. No. 5,824,467), as well as methods for creating predictive models of responsiveness to a particular treatment (U.S. Pat. No. 6,087,090).
IGFBP-3 is a master regulator of cellular function and viability. As the primary carrier of IGFs in the circulation, it plays a central role in sequestering, delivering and releasing IGFs to target tissues in response to physiological parameters such as nutrition, trauma, and pregnancy. IGFs, in turn, modulate cell growth, survival and differentiation, additionally; IGFBP-3 can sensitize selected target cells to apoptosis in an IGF-independent manner The mechanisms by which it accomplishes the latter class of effects is not well understood but appears to involve selective cell internalization mechanisms and vesicular transport to specific cellular compartments (such as the nucleus, where it may interact with transcriptional elements) that is at least partially dependent on transferrin receptor, integrins and caveolin.
The inventor has previously disclosed certain IGFBP-derived peptides known as “MBD” peptides (U.S. patent application publication nos. 2003/0059430, 2003/0161829, and 2003/0224990). These peptides have a number of properties, which are distinct from the IGF-binding properties of IGFBPs, that make them useful as therapeutic agents. MBD peptides are internalized some cells, and the peptides can be used as cell internalization signals to direct the uptake of molecules joined to the MBD peptides (such as proteins fused to the MBD peptide).
Combination treatments are increasingly being viewed as appropriate strategic options for designed interventions in complex disease conditions such as cancer, metabolic diseases, vascular diseases and neurodegenerative conditions. For example, the use of combination pills containing two different agents to treat the same condition (e.g. metformin plus a thiazolidinedione to treat diabetes, a statin plus a fibrate to treat hypercholesterolemia) is on the rise. It is therefore appropriate to envisage combination treatments that include moieties such as MBD in combination with other agents such as other peptides, antibodies, nucleic acids, chemotherapeutic agents and dietary supplements. Combinations may take the form of covalent extensions to the MBD peptide sequence, other types of conjugates, or co-administration of agents simultaneously or by staggering the treatments i.e. administration at alternating times.
Humanin (HN) is a novel neuroprotective factor that consists of 24 amino acid residues. HN suppresses neuronal cell death caused by Alzheimer's disease (AD)-specific insults, including both amyloid-beta (betaAbeta) peptides and familial AD-causative genes. Cerebrovascular smooth muscle cells are also protected from Abeta toxicity by HN, suggesting that HN affects both neuronal and non-neuronal cells when they are exposed to AD-related cytotoxicity. HN peptide exerts a neuroprotective effect through the cell surface via putative receptors (Nishimoto I et al [2004] Trends Mol Med 10: 102-105). Humanin is also a neuroprotective agent against stroke (Xu X et al [2006] Stroke 37: 2613-2619). As has previously been demonstrated, it is possible to generate both single-residue variants of humanin with altered biological activity and peptide fusions of humanin to other moieties (Tajima H et al [2005] J. Neurosci Res. 79 (5): 714-723; Chiba T et al. [2005] J. Neurosci. 25: 10252-10261). This indicates the feasibility of combining humanin peptide sequences with, for example, MBD-based therapeutic peptides or, alternatively, the therapeutic segments of previously described MBD-linked therapeutic peptides. The solution structures of both native humanin and its S14G variant have been described (Benaki D et al [2005] Biochem Biophys Res Comm 329: 152-160; Benaki D et al [2006] Biochem Biophys Res Comm 349: 634-642) thereby potentially facilitating the design of mutant or derivative sequences. The amino acid sequence of humanin is MAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 1) and the amino acid sequence of the variant is MAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO:2). Humanin binds a C-terminal domain of IGFBP-3 (Ikonen M et al [2003] Proc Nat Acad Sci. 100: 13042-13047). The binding of Zinc(II) to humanin was recently described (Armas A et al [2006] J. Inorg Biochem 100: 1672-1678). Therefore humanin may be considered a metal-binding therapeutic peptide.
Potentially therapeutic peptide sequences have been disclosed in the scientific literature. Many of these require cell internalization for action, which limits their in vivo utility without an appropriate delivery system. Peptide sequences that bind and possibly inhibit MDM2 (Picksley S M et al [1994] Oncogene. 9: 2523-2529), protein kinase C-beta (Ron D et al [1995] J Biol Chem. 270: 24180-24187), p38 MAP kinase (Barsyte-Lovejoy D et al [2002] J Biol Chem. 277: 9896-9903), DOK1 (Ling Y et al [2005] J Biol Chem. 280: 3151-3158), NF-kappa-B nuclear localization complex (Lin Y Z et al [1995] J Biol Chem. 270: 14255-14258), IKK complex (May M J et al [2000] Science. 289:1550-1554) and calcineurin (Aramburu J et al [1999] Science. 285: 2129-33) have been described.
We have shown that MBD peptide-mediated delivery of bioactive molecules in vivo can be applied to disease processes such as cancer (Huq A, et al [2009] Anti-Cancer Drugs 20: 21-31) and diabetes, as described above. Nephrilin, a peptide containing the MBD scaffold, is bioactive in reducing albuminuria in diabetic mice. Nephrilin was designed to interfere with mTORC2 complex and has been shown to disrupt the association of IRS proteins with Rictor. Similar approaches may be used to disrupt mTORC2 and IRS protein activity in human disease by competing the physical interaction of Rictor with obligate cofactors such as PRR5/Protor and Sin1/MIP1. The competing molecule may be a cell-penetrating peptide, protein, antibody or nucleic acid, or a small chemical molecule. In this work we describe in vitro assay systems that facilitate rapid screening of candidate molecules for such a purpose. Any metabolic, systemic, degenerative, or inflammatory disease process may be a candidate for interventions using such molecules.
A noteworthy observation from this work is the previously undocumented elevation of SGK1 in the spleen tissue of db/db mice. Post-hoc subgroup analysis of control animals showed a small (˜10%) but significant exacerbation of kidney disease marker elevation in the subgroup with higher spleen SGK1. Nephrilin, but not anephril, was able to reduce spleen SGK1 significantly. A possible subject for future study would be to see if SGK1 is elevated in the circulating leukocytes of animals that exhibit elevated spleen SGK1, and whether a diagnostic possibility exists for predicting the severity of disease based on SGK1 levels in white blood cells.
The central role played by mTORC2 in regulating diseases of aging has not been previously documented. Nephrilin, an inhibitor of the binding of Rictor—the canonical component of mTORC2 complex—to its binding partners or cofactors such as Protor/PRR5, Sin1 and IRS proteins, is the only specific inhibitor of its class described to date. The inventor has shown, for the first time, that nephrilin can reverse disease processes relating to complications of diabetes and hypertension; acute kidney injury from rhabdomyolysis or xenotoxic stress with platinum compounds such as cisplatin and carboplatin or aminoglycoside antibiotics such as gentamycin; and cancer metastasis. These results implicate mTORC2 as a central regulator of diseases of aging. Fundamental common mechanisms suggested for the gamut of diseases of aging—the so-called diseases of western civilization—include oxidative stress [Pinton P and Rizzuto R (2008) Cell Cycle. 7(3): 304-308], loss of circadian circuitry [Uchida Y et al (2010) Biol. Pharm. Bull. 33(4) 535-544], loss of selective protein turnover mechanisms [Hussain S et al (2009) Cell Cycle 8:11, 1688-1697], and the epithelial-mesenchymal transition, EMT [Slattery C et al (2005) American Journal of Pathology, 167(2): 395-407]. The inventor has shown that biochemical signatures associated with each of these pathways can be reversed by treatment with nephrilin and has demonstrated, for the first time, that specific inhibition of mTORC2 (which should not be confused with the much better-understood rapamycin-sensitive complex mTORC1) may be the key to controlling diseases of aging. Thus, therapeutic agents that disrupt binding of Rictor (the canonical component mTORC2) to its binding partners are of particular interest in the treatment of metabolic and cardiovascular diseases, especially those characterized by some underlying combination of insulin resistance, hyperglycemia, hypertension and hyperlipidemia; cancer progression and metastasis; acute kidney injury (AKI) in critical care settings (including sepsis, systemic inflammatory conditions such as shock, post-operative stress such as after cardiopulmonary bypass or transplant, burns, pancreatitis, rhabdomyolysis, xenobiotic stresses caused by cocaine, alcohol, aminoglycoside antibiotics, antiviral compounds or platinum compounds; neurodegenerative diseases such as Parkinson's Alzheimer's, Huntington's and ALS/Lou Gehrig's disease; ototoxicities; autoimmune conditions such as lupus erythematosus and multiple sclerosis; genetic diseases such as cystinosis, Fanconi's and other conditions affecting mitochondrial respiration; pulmonary diseases, especially COPD and asthma; ocular diseases such as cataracts and retinopathies, especially diabetic complications; and liver diseases, including chronic viral infections such as hepatitis. These disease states are now increasingly viewed as secondary to chronic inflammatory conditions that may, in turn, relate to oxidative stress. A correlation between oxidative stress and processes of aging may explain the rising incidence of these diseases as a direct consequence of an aging population.
A key regulator of oxidative damage and aging is the adapter protein p66shc. Activation of this molecule by phosphorylation at serine 36 leads to mitochodrial translocation and increased production of free oxygen radicals. P66shc gene knockout mice live significantly longer and are protected from many of the diseases of aging listed above [Pinton P and Rizzuto R (2008) Cell Cycle. 7(3): 304-308]. The inventor has shown for the first time, that mTORC2 directly regulates the activation of protein kinase C beta-II (PKC-beta-II) by phosphorylation at threonine 641. PKC-beta was shown to be the activator of p66shc by phosphorylation of S36 [Pinton, et al. (2007) Science 315: 659-663]. Nephrilin reverses both PKC-beta-II-T641 and p66shc-S36 phosphorylation events.
A recently recognized histological consequence of cellular stress is the formation of microscopically visible punctate structures in the nuclei of stressed cells [Bart J. et al (2008) Cytometry 73A: 816-824]. The inventor has shown, in diseased hypertensive animals, the presence of such structures in kidney cells by immunohistochemical staining. The incidence of such structures is much reduced in animals treated with the mTORC2 inhibitor, nephrilin.
Epithelial cells of renal proximal tubules (PTECs) are known to be exquisitely sensitive to p66shc-mediated oxidative stress [Sun L et al (2010) Am J Physiol Renal Physiol. 299(5): F1014-F1025]. Damage to PTECs can be monitored by measuring albumin or lipocalin-2/NGAL in urine by using commercially available kits. In many of the proinflammatory disease conditions listed above, elevated levels of NGAL or albumin have been documented. This is especially true of AKI settings such as those encountered in patients with burns, hypoperfusion, pancreatitis and sepsis [Cruz D et al (2010) Intensive Care Med 36:444-451]. In AKI, moreover, a proinflammatory condition reminiscent of human systemic inflammatory states encountered in critical care settings, as enumerated above, can be generated in experimental animals by placing artificial stress on kidneys, such as in rhabdomyolysis and gentamycin models [Zager R et al (2006) Am J Physiol Renal Physiol 291:F546-F556]. The inventor has shown, for the first time, that this type of proinflammatory state is regulated by mTORC2 and can be successfully treated by an inhibitor of mTORC2, nephrilin.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.